Bio-Engineered Liver Organoids for Drug Screening and Cell Replacement Therapy

Engineered tissue represents a great promise for treating liver diseases. Differentiated liver organoids have shown to be an interesting alternative to the native liver-derived hepatocytes. Controlling the progression of differentiation in live cells could have important implications for obtaining fully functional terminally differentiated liver organoids. In order to monitor the progression of differentiating organoids consisting of HLA-Class II knockout hiPSCs interlaced with endothelial cells toward functional liver organoids, we have complemented the conventional real-time PCR measurements of hepatic markers and coagulation factors with the noninvasive Raman spectroscopy.

Methods

Generation of human embryoid bodies (hEBs) using HLA-Class II knockout hiPSCs interlaced with endothelial cells was achieved using ROCKi/Spin-free technology we developed earlier. Raman spectra were collected in live organoids using a Raman spectroscopy system consisting of a 785 nm excitation diode laser and a portable Raman spectrometer. We also performed clinically relevant assays for measuring hepatic function ex vivo.

Results

Changes in Raman spectra of organoids over the course of 16 days, spanning four stages of differentiation, exhibited peaks that can be associated with CER1, AFP, and creatinine (Fig. 1a-c). Gene expression of hepatic markers and coagulation factors were detected by real-time PCR, showing the maturation of liver organoids (Fig. 1d).

Conclusions

Real-time monitoring of differentiating hiPSC organoids represents an excellent tool to assess the differentiation progress in live cells. Our results demonstrated the potential of Raman spectroscopy to complement conventional techniques providing information on hepatic differentiation noninvasively. This technique could be important for successful clinical translation.